TRANSGENIC MODELS OF RESPIRATORY CONTROL

呼吸控制的转基因模型

• 批准号: 6537366
• 负责人: DAVID E MILLHORN
• 金额: $ 26.28万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-10 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537366
• 关键词: PC12 cells; animal genetic material tag; biological models; calcium transporting ATPase; calmodulin dependent protein kinase; carotid body; cell membrane; endoplasmic reticulum; enzyme activity; genetically modified animals; hypercapnia; laboratory mouse; model design /development; neuroregulation; phosphoprotein phosphatase; pulmonary respiration; respiratory hypoxia; sarcoplasmic reticulum; tissue /cell culture

DESCRIPTION: The primary compensation for arterial hypoxemia is hyperventilation which is mediated by the O2-sensitive cells in the carotid body. The carotid body O2 chemoreceptors play a critical role in the maintenance of O2-homeostasis. The mechanisms by which the O2-sensitive (type I) cells in the carotid body detect a reduction in O2 tension and transduce this signal into the appropriate cellular responses leading to hyperventilation remains, for the most part, unknown. It is known, however, that type I cells express an O2-sensitive K channel that is inhibited by reduced O2 tension which, in turn, causes membrane depolarization and an increase in intracellular free Ca2+. We have shown that hypoxia-induced expression of the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, requires an increase in cytosolic Ca2+ and activation of calmodulin (CaM) in type I cells and in PC12 cells, an O2-sensitive cell line. We also found that neutralization of CaM in catecholamine cells in transgenic mice prevents hyperventilation and increased carotid body activity during hypoxia. In the proposed studies we shall investigate the molecular basis by which the Ca2+/CaM signal transduction system regulates carotid body function in transgenic mice during hypoxia. We hypothesize that an increase in intracellular free Ca2+ and activation of Ca2+/CaM target enzymes (CaM-KI, CaM-KII and CaIN) are involved in this critical process. We further hypothesize that calcium pumps (SERCA2, SERCA3 and PMCA2) play a major role in regulating the level of cytosolic Ca2+ during hypoxia. This is an important function which couples the level of intracellular free Ca2+ with the prevailing hypoxic stimulus. The Specific Aims of the proposed research are: 1) Determine the role of CaM-activated kinases and phosphatases in mediating the cellular responses to hypoxia in wild-type and genetically modified PC12 cells, 2) Determine the role of CaM activated-kinases and phosphatases in mediating the carotid body and ventilatory response to hypoxia in transgenic mice in which these pathways have been neutralized by a novel genetic approach, and 3) Determine the role of the CaM-sensitive plasma membrane and the sarco(endo)plasmic Ca2+-ATPases in mediating the cellular response to hypoxia. Studies are performed in the O2-sensitive PC12 cells, type I cells, transgenic mice and gene knockout mice. Findings from the proposed research should provide much needed information concerning the cellular and molecular basis of O2 chemosensitivity.

描述：动脉低氧血症的主要代偿是 过度换气，由颈动脉中的O2敏感细胞介导 身体 颈动脉体O2化学感受器在动脉粥样硬化中起关键作用。 维持O2-稳态。 O2-敏感性的机制 (type I）颈动脉体中的细胞检测到O2张力的降低， 将此信号转化为适当的细胞反应， 换气过度在很大程度上仍然是未知的。 然而，众所周知， I型细胞表达O2敏感性K通道， 降低O2张力，这反过来又导致膜去极化， 细胞内游离Ca ~（2+）增加。 我们已经证明，缺氧诱导的 编码酪氨酸羟化酶（TH）的基因的表达， 多巴胺生物合成中的限速酶，需要增加 在I型细胞中胞浆Ca 2+和钙调素（CaM）的活化中， PC 12细胞，一种O2敏感细胞系。 我们还发现， 转基因小鼠中的儿茶酚胺细胞中的CaM防止过度通气， 缺氧时颈动脉体活性增加。 在拟议的研究中， 将研究Ca 2 +/CaM信号的分子基础 转导系统调节转基因小鼠颈动脉体功能 在缺氧的时候。 我们假设细胞内游离Ca 2+的增加 和Ca 2 +/CaM靶酶（CaM-KI、CaM-KII和CaIN）的活化是 参与到这一关键过程中。 我们进一步假设钙 泵（SERCA 2、SERCA 3和PMCA 2）在调节水平方面起主要作用 缺氧时胞浆Ca ~（2+）的变化。 这是一个重要的功能， 将细胞内游离Ca 2+水平与主要的缺氧 刺激。 本研究的具体目标是：1）确定 钙调素激活的激酶和磷酸酶在介导细胞凋亡中的作用 野生型和遗传修饰的PC 12细胞对缺氧的反应，2） 确定CaM激活的激酶和磷酸酶在介导 转基因小鼠颈动脉体及缺氧反应 这些途径已经被一种新的遗传方法中和， 3)确定钙调素敏感的质膜和细胞膜的作用。 肌浆内Ca ~（2+）-ATP酶介导细胞对 缺氧 在O2敏感的PC 12细胞中进行研究，I型 细胞、转基因小鼠和基因敲除小鼠。 建议的调查结果 研究应提供急需的信息， O2化学敏感性的分子基础。